Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two genes (gdrAB) of Klebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol. 181, 4110-4113). Recombinant GdrA and GdrB proteins formed a tight complex of (GdrA)(2)(GdrB)(2), which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O(2)-inactivated glycerol dehydratases as well as activated the enzyme-cyanocobalamin complex in vitro in the presence of ATP, Mg(2+), and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins in the presence of ATP and Mg(2+) through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme with the factor dissociated upon incubation with ATP, forming functional apoenzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme-factor complex. We propose that the glycerol dehydratase-reactivating factor is a molecular chaperone that participates in reactivation of the inactivated enzymes.

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase.

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.

Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of Klebsiella pneumoniae, the inactivated enzyme is reactivated in the presence of ATP, Mg2+, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them gdrA and gdrB. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant Escherichia coli cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.

Initiation of cyclin B degradation by the 26S proteasome upon egg activation.

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.

Isolation and characterization of the cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis.

A cDNA clone encoding the complete tilapia (a teleost fish, Oreochromis niloticus) cytochrome P450 aromatase (P450arom) was isolated from an ovarian follicle cDNA library. The deduced amino acid sequence (522 amino acid residues) had 72.2% and 59.5% homology with rainbow trout and catfish P450arom respectively, and about 50% homology with mammalian and avian P450arom. Expression of this cDNA in COS-7 cells produced a protein that converted exogenous testosterone to estrogens. Northern blots using a tilapia P450arom cDNA fragment and Western blots using an antiserum against a tilapia P450arom polypeptide fragment revealed a single P450arom mRNA (2.6 kb) and a single protein (59 kDa) in tilapia ovarian tissue respectively. These analyses also revealed that the levels of both P450arom mRNA and protein were low in early vitellogenic follicles, increased in midvitellogenic follicles, and declined to non-detectable levels in post-vitellogenic follicles. Changes in the ability of follicles to convert exogenous testosterone to estrogens (aromatase activity) were similar to those of P450arom mRNA and protein. These observations indicated that the capacity of tilapia ovarian follicles to synthesize estradiol-17 beta is closely related to the contents of P450arom mRNA and protein within them.

Molecular cloning and immunological analysis of goldfish cyclin A during oocyte maturation.

Cyclin A belongs to a family of proteins involved in the regulation of the eukaryotic cell cycle. Although cyclin A is thought to be involved in the regulation of both S and M phase, its exact role in the cell cycle, especially in the meiotic cycle (oocyte maturation), is uncertain. We isolated cyclin A cDNA clones from a goldfish oocyte cDNA library. Monoclonal antibody raised against bacterially produced goldfish cyclin A recognized a 47-kDa protein that disappeared after egg activation. Unlike goldfish cyclin B, which is absent in immature oocytes, cyclin A was already present in immature oocytes and its protein level did not change remarkably during oocyte maturation. These results differ from the finding in Xenopus, in which cyclin A is absent, but cyclin B is present, in immature oocytes. Goldfish cyclin A was associated with cdc2 kinase in mature oocytes, but not with cdk2. Recombinant cyclin A bound to and activated cdc2 in a cell-free system, but cyclin A and cdk2 binding was not observed. The kinase activity of cyclin A-cdc2 was undetectable in immature oocytes and first appeared at about the time of germinal vesicle breakdown (GVBD). In contrast to the cyclin B-cdc2 activity that corresponded to the occurrence of GVBD, cyclin A-cdc2 activity increased only slightly until GVBD was completed and increased drastically after the completion of the first meiotic division. Furthermore, microinjection of cyclin A mRNA into immature oocytes did not cause GVBD; however, microinjection of cyclin B mRNA did. These results suggest that cyclin A-cdc2 kinase and cyclin B-cdc2 kinase play different roles in controlling oocyte maturation. The roles of cyclin A in the rapid activation of cyclin B-cdc2 kinase at meiosis I and II transition and in the maintenance of high maturation-promoting factor activity in mature unfertilized eggs are discussed.

Molecular mechanisms of the activation of maturation-promoting factor during goldfish oocyte maturation.

Oocyte maturation is triggered by the activation in the oocyte cytoplasm of maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). Immature goldfish oocytes contain only inactive monomeric 35-kDa cdc2 and do not stockpile cyclin B. In maturing oocytes, activation of cdc2 is associated with its Thr161 phosphorylation and mobility shift on SDS-PAGE from 35 to 34 kDa after binding to cyclin B. Using mutant cdc2, we show that Thr161 phosphorylation is required for both the downward shift and the kinase activation. Since cdc2 Tyr15 is not phosphorylated after binding to cyclin B, it does not require dephosphorylation. This situation is obviously different from that in immature Xenopus oocytes, in which the cdc2-cyclin B complex preexists with cdc2 phosphorylated on both Tyr15 and Thr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25 phosphatase for MPF activation. These results indicate that these species employ different mechanisms of MPF activation during oocyte maturation, although the final molecular structure of the active MPF (cdc2 bound to cyclin B and phosphorylated on Thr161) is identical.

Different responsiveness of the chorda tympani and glossopharyngeal nerves to L-lysine in mice.

Differential taste responsiveness and functional role of the two taste nerves, the chorda tympani (CT) and the glossopharyngeal (GL), were studied in mice by examining neural and behavioral responses to an essential amino acid, L-lysine (Lys). Relative responses to Lys were larger in the GL than in the CT nerve. The neural threshold for the Lys response was about 2.5 log units lower in the GL (about 1.0 microM) than in the CT nerve (about 300 microM). An analysis of concentration-response relationships suggests a possibility that there are two different receptors (high and low affinity types) for Lys showing different dissociation constants. The posterior tongue region possesses both types, while the anterior region possesses only the low affinity type. Behavioral aversion threshold for Lys in intact mice, measured by use of a single bottle test, was about 1.0 microM. This threshold was the same as its neural threshold in the GL nerve. Animals whose bilateral GL nerves were sectioned showed a higher aversion threshold (about 300 microM) which was the same as the neural threshold in the CT nerve. An aversion conditioned to Lys significantly generalized to L-arginine in the intact and CT-denervated mice, and L-arginine and L-histidine in the GL-denervated mice, but the generalization pattern across various taste stimuli including the four basic taste stimuli (NaCl, HCl, quinine HCl and sucrose) did not prominently differ among the intact, the GL-denervated and CT-denervated mice. These results suggest that taste sensitivity to Lys is higher in the GL than in the CT nerve, but taste quality information for Lys conveyed by two taste nerves is not largely different.

Glossopharyngeal denervation alters responses to nutrients and toxic substances.

Functional roles of the glossopharyngeal (GL) nerve on food and fluid intake were studied by examining effects of the GL denervation on two biologically different activities induced by specific diets using mice and rats. First, we examined whether GL section alters the acceptability of a bitter tasting essential amino acid, L-lysine (Lys), by Lys-deficiency in mice. The aversion threshold for Lys, normally about 3 uM in mice, increased to about 300 uM when mice were fed the Lys-deficient diet for 10 days. This increase of the Lys aversion threshold (increase of acceptability for Lys) by Lys-deficiency was also evident in mice with the chorda tympani denervation but was not observed in mice with the GL denervation. Next, we examined whether GL section alters the induction of a salivary protein, cystatin S (a cysteine proteinase inhibitor), by a diet containing papain (a cysteine proteinase) in rats. GL denervation largely inhibited the induction of cystatin S in the rat submandibular glands by papain. These results collectively suggest that chemosensory information conveyed by the GL nerve plays important roles on recognition of both nutrient and toxic compounds in the diet and induction of biological responses that protect the animal from both nutritional deficiency and exogenous toxic compounds.

Differential taste responses of mouse chorda tympani and glossopharyngeal nerves to sugars and amino acids.

The differential taste responses of the chorda tympani (CT) and the glossopharyngeal (GL) nerves in preweanling and adult mice were examined by comparing magnitudes of responses to six sugars and 10 amino acids. The results indicate that for sugars the responses of the CT nerve are greater than those of the GL nerve while for umami and essential amino acids the responses of the GL nerve are greater than those of the CT nerve. Such differential taste responses of the CT and GL nerves does not prominently change during development.

Behavior of the components of maturation-promoting factor, cdc2 kinase and cyclin B, during oocyte maturation of goldfish.

We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.

Enhancement of murine gustatory neural responses to D-amino acids by saccharin.

Taste enhancing effects of sodium saccharin (Sac) on responses to particular sweet-tasting D-amino acids were found during the recording of mouse chorda tympani nerve responses to various taste stimuli in C57BL and BALB strains. In both strains, responses to D-tryptophan and D-histidine significantly increased (167.7-216.7% of control) after the stimulation with Sac as compared with those applied before Sac. In C57BL mice, the enhancement of Sac was also observed in response to D-phenylalanine (262.5% of control), but this was not the case for BALB mice, suggesting a prominent strain difference in response to D-phenylalanine, as shown previously. Responses to other sweet-tasting D- and L-amino acids and sugars were not enhanced by Sac. Enhancement of responses to these D-amino acids by Sac was also evident when responses to a mixture of D-amino acids and Sac were compared with the sum of responses to each component, although in this response analysis, the calculated magnitude of enhancement generally become smaller (135.7-180.5% of the sum) and enhancement of D-histidine responses disappeared. Except for Sac, various sweet-tasting amino acids and sugars and NaCl also tested showed no enhancing effect on D-phenylalanine responses in C57BL mice. Sac and D-amino acids, to which responses were enhanced by Sac, possess some common molecular features, namely ring structures. This structural similarity probably relates to the occurrence of the enhancement at the receptor sites.

Early developmental change in bitter taste responses in human infants.

Human newborns (birth-6 days) and older infants (14-180 days) were allowed to ingest both urea (0.12-0.24 M) in a mildly sweet diluent and the diluent alone, and multiple measures of responsivity were obtained (relative intake, sucking behavior, and hedonic ratings based on facial expressions and body movements). For newborns, there was no indication of rejection of urea relative to the diluent in measures of intake or sucking behavior; rather, their responses were predominantly controlled by the order of presentation of the two tastes. In contrast, older infants tended to reject all concentrations of urea according to these measures. Hedonic ratings provided an indication of limited rejection of the bitter taste by newborns, but older infants were still found to respond more consistently. These data suggest there is an early developmental change in bitter taste perception.

Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2.

This paper reports the nucleotide and predicted amino acid sequences of the goldfish cdk2, a cognate variant of the cell cycle regulator cdc2. The predicted protein sequence shows strong homology to the other known cdk2 (88% for Xenopus and 90% for human). A monoclonal antibody against the C-terminal sequence of goldfish cdk2 recognized a 34-kDa protein in extracts from various goldfish tissues. The protein level was high in such tissues as testis and ovary containing actively dividing cells. Protein cdk2 binds to p13sucl, the fission yeast suc1+ gene product, but not to cyclin B, with which cdc2 forms a complex. The kinase activity of cdk2 increased 30-fold when oocytes matured, although its protein level did not remarkably change. Anti-cdk2 immunoprecipitates from 32P-labeled mature oocyte extracts contained a 47-kDa protein, which was not recognized by either anti-cyclin A or anti-cyclin B antibody, indicating complex formation of cdk2 with a protein other than cyclins A or B.

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein.

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an intraluminal mediator in rat pancreatic enzyme secretion: reconstitution of the pancreatic response with dietary protein, trypsin and the monitor peptide.

New evidence has been obtained suggesting that the "monitor" peptide is an essential intraluminal mediator in the stimulation of pancreatic enzyme secretion in response to protein intake in rats. Experiments were performed in vivo using a mixture of 50 mg of ovalbumin, alpha-lactalbumin or casein, 2 micrograms of purified protease-sensitive, cholecystokinin-releasing monitor peptide and 1 mg of porcine trypsin which was infused by cannula into the duodenum of atropine-treated rats. The small intestine had previously been washed with bicarbonate to eliminate proteases and the pancreatic juice was diverted. The amount of trypsin secreted in 2 h was comparable to that of rats in which the pancreatic juice was returned into the duodenum. However, in the presence of a monitor peptide--specific antibody which recognizes the N-terminal region of the peptide, the monitor peptide did not induce any pancreatic response. Therefore, the characteristic pattern of pancreatic enzyme secretion in response to protein intake can be reproduced by infusing only three components--dietary proteins, porcine trypsin and the purified monitor peptide.

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms.

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus.

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.

Some more speract derivatives associated with eggs of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina.

1. Fourteen peptides were isolated from the egg jelly of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina and their amino acid sequences were determined. 2. The peptides stimulated H. pulcherrimus sperm respiration one half-maximally at about 8-60 pM. 3. Addition of speract to intact spermatozoa of P. depressus, H. pulcherrimus and A. crassispina resulted in the appearance of a newly stained protein (Mr 128,000 for P. depressus, Mr 128,000 for H. pulcherrimus and Mr 131,000 for A. crassispina) on sodium dodecyl sulfate-polyacrylamide gels.

Separation, amino-terminal sequence and cell-free synthesis of the smallest subunit of sweet potato cytochrome c oxidase.

The smallest subunit (V) of sweet potato cytochrome c oxidase was separated into three polypeptides, Va, Vb and Vc with different molecular masses (7.4 kDa, 6.8 kDa and 6.2 kDa respectively) by highly resolving sodium dodecylsulfate polyacrylamide gel electrophoresis. Antibody against subunit V reacted specifically with the polypeptide Vc. When polyadenylated mRNA from sweet potato root tissue was translated in a wheat germ cell-free system, the smallest subunit (Vc) of the polypeptides was synthesized to the same size as the mature form, which suggests that the mature subunit retains the signal for import into mitochondria. Within the N-terminal first 25 amino acids there is a stretch of 16 non-polar residues, periodically linked by basic residues, which might form an amphiphilic helix as the targeting signal.